U.S. patent application number 12/997411 was filed with the patent office on 2011-06-16 for conjugated vi saccharides.
Invention is credited to Francesco Berti, Paolo Costantino, Francesca Micoli.
Application Number | 20110142876 12/997411 |
Document ID | / |
Family ID | 39672273 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110142876 |
Kind Code |
A1 |
Micoli; Francesca ; et
al. |
June 16, 2011 |
CONJUGATED VI SACCHARIDES
Abstract
Two Vi conjugates have been prepared by carbodiimide-mediated
synthesis, using adipic acid dihydrazide derivatized CRM.sub.197 (a
non-toxic variant of diphtheria toxin) and tetanus toxoid, as
carrier proteins.
Inventors: |
Micoli; Francesca; (Siena,
IT) ; Costantino; Paolo; (Colle Val D'Elsa, IT)
; Berti; Francesco; (Colle Val D'Elsa, IT) |
Family ID: |
39672273 |
Appl. No.: |
12/997411 |
Filed: |
June 12, 2009 |
PCT Filed: |
June 12, 2009 |
PCT NO: |
PCT/IB2009/006285 |
371 Date: |
February 25, 2011 |
Current U.S.
Class: |
424/197.11 ;
514/20.9; 530/395; 536/55.1 |
Current CPC
Class: |
A61K 39/0275 20130101;
A61K 47/6415 20170801; A61P 37/04 20180101; C07K 14/195 20130101;
A61P 43/00 20180101; A61K 47/646 20170801 |
Class at
Publication: |
424/197.11 ;
530/395; 514/20.9; 536/55.1 |
International
Class: |
A61K 39/385 20060101
A61K039/385; C07K 2/00 20060101 C07K002/00; A61K 38/02 20060101
A61K038/02; C08B 37/00 20060101 C08B037/00; A61P 37/04 20060101
A61P037/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2008 |
GB |
0810894.6 |
Claims
1. A method for producing a conjugate of Vi comprising the steps
of: a) simultaneously combining a linker, a carbodiimide and a
carrier protein; b) removing any excess linker from the product of
step a); c) reacting Vi with a carbodiimide; and d) reacting the
product of step b) with the product of step c).
2. A method according to claim 1, wherein the excess linker is
removed by dialysis.
3. A method according to claim 1, wherein the carrier protein is
either CRM 197 or tetanus toxoid.
4. A method according to claim 1, wherein the carbodiimide is
1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC).
5. A method according to claim 1, wherein the linker is adipic acid
dihydrazide.
6. A conjugate comprising Vi linked to CRM 197.
7. A conjugate according to claim 6, wherein the Vi is linked to
the CRM 197 by an adipic acid dihydrazide (ADH) linker.
8. A pharmaceutical composition comprising the conjugate of claim 6
in combination with a pharmaceutically acceptable carrier.
9. A pharmaceutical composition according to claim 8, wherein the
composition is unadjuvanted.
10. A pharmaceutical composition according to claim 8, wherein the
composition further comprises an adjuvant.
11. The composition of claim 8, comprising saline.
12. The composition of claim 8, wherein a unit dose of the
composition includes 5 .mu.g of Vi saccharide.
13. A method for raising an immune response in a mammal, comprising
administering the conjugate of claim 6 to the mammal.
14. A method for derivatising a Vi saccharide, comprising reacting
the saccharide with a carbodiimide at a Vi:carbodiimide molar ratio
of >3:1.
15. The method of claim 11, wherein the ratio is >5:1.
16. The method of claim 12, wherein the ratio is >9:1.
17. The method of claim 13, wherein the carbodiimide is EDAC.
18. A method for raising an immune response in a mammal, comprising
administering the pharmaceutical composition of claim 8 to the
mammal.
Description
[0001] This application claims the benefit of United Kingdom patent
application 0810894.6, filed 13 Jun. 2008, the complete contents of
which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The invention relates to vaccines, more particularly those
against typhoid fever.
BACKGROUND ART
[0003] Typhoid fever is a common serious disease in many parts of
the world. Purified capsular polysaccharide from Salmonella typhi
(Vi) is used as a vaccine, providing about 70% protection against
typhoid fever in individuals 5- to 45-years-old. However, the
vaccine is unable to establish immunological memory and is
ineffective in infants or toddlers [1]. A conjugate vaccine of Vi
coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A
(Vi-rEPA) gave a booster response in young children and was highly
efficacious [2].
[0004] It is an object of the invention to provide new processes
for the production of Vi conjugate vaccines that may be employed on
an industrial scale.
DISCLOSURE OF THE INVENTION
[0005] The inventors have devised a new method for manufacturing
Vi-conjugates and have also produced a new conjugate comprising Vi
coupled to CRM.sub.197.
[0006] A first aspect of the invention provides a method for the
preparation of a Vi conjugate. A linker, such as adipic acid
dihydrazide (ADH), and a carbodiimide, such as
1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC), are
simultaneously added to a solution containing a carrier protein,
such as CRM.sub.197 or tetanus toxoid (TT), to give a derivatised
carrier protein.
[0007] A buffer, such as 2-(N-morpholino)ethanesulfonic acid (MES),
may be added to the solution containing the carrier protein prior
to the addition of ADH and EDAC. The weight ratio of the
carbodiimide to the protein is typically 0.1 to 0.15, as higher
carbodiimide/protein ratios can cause aggregate formation.
[0008] Following the derivatisation of the carrier protein, any
excess linker (e.g. ADH) is removed by, for example, dialysis or
tangential flow filtration (TFF).
[0009] Vi is also activated with a carbodiimide and is subsequently
combined with the derivatised carrier protein. For Vi activation,
various ratios of Vi and carbodiimide can be used. A 1:1 molar
ratio (COOH groups of Vi to carbodiimide) can be used, but to
reduce the amount of residual unconjugated carbodiimide derivatives
(e.g. ureas such as EDU; N-ethyl-N'-(3-dimethylaminopropyl)urea, a
soluble reaction product of EDAC coupling) higher ratios can be
used i.e. with a molar excess of Vi e.g. >1.5:1 and ideally
.gtoreq.3:1, such as 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or higher.
Ratios up to 200:1 might be used. These ratios are higher than the
ones used in reference 3. Vi activation can be performed at room
temperature e.g. in about 2 minutes.
[0010] Thus, the method comprises the steps of: [0011] a)
Simultaneously combining a linker, a carbodiimide and a carrier
protein. [0012] b) Reacting Vi with a carbodiimide [0013] c)
Reacting the product of step a) with the product of step b).
[0014] Steps a) and b) may be performed in any order.
[0015] After step a), but before step c), any excess linker may be
removed.
[0016] This aspect of the invention also provides a method for
preparing a Vi-CRM.sub.197 conjugate, wherein Vi is combined with
derivatised CRM.sub.197. The method comprises the steps of: [0017]
a) Reacting Vi with a carbodiimide [0018] b) Reacting the product
of step a) with derivatised CRM.sub.197.
[0019] This aspect of the invention also provides a method for
preparing a Vi-CRM.sub.197 conjugate, comprising the step of:
[0020] a) Reacting activated Vi with derivatised CRM.sub.197.
[0021] Reference 3 describes a process for preparing conjugates of
Vi with rEPA and bovine serum albumin (BSA) by
carbodiimide-mediated synthesis with ADH as the linker. However,
this process does not involve the simultaneous combination of
rEPA/BSA, ADH and EDAC. Rather, rEPA/BSA and ADH are combined and
mixed prior to the addition of EDAC.
[0022] A second aspect of the invention provides a Vi-CRM.sub.197
conjugate. This conjugate may be prepared by the above method, or
may be obtained by other means.
Vi Saccharide
[0023] Vi is the capsular saccharide of Salmonella typhi
(previously classified as a species itself, but now referred to as
the typhi serovar of S. enterica). Vi may also be found in other
serovars of Salmonella (such as S. enterica serovar paratyphi C or
serovar dublin) and in other bacteria, such as Citrobacter (e.g. C.
freundii and C. youngae). The Vi polysaccharide is a linear
homopolymer of a hexosaminuronic acid,
.alpha.-1,4-N-acetylgalactos-aminouronic acid, which is 60-90%
acetylated at the C-3 position [4-9]. The O-acetyl substitution on
Vi is a factor in its ability to elicit a protective immune
response [10]. The immunogenicity of Vi is closely related to its
degree of O-acetylation. Partial de-O-acetylation can slightly
increase immunogenicity; complete de-O-acetylation eliminates the
immunogenicity of Vi [11].
[0024] The Vi saccharide used in the present invention may be
chemically modified relative to the capsular saccharide as found in
nature. For example, the Vi saccharide may be partially
de-O-acetylated, de-N-acetylated (partially or fully),
N-propionated (partially or fully), etc. De-acetylation may occur
before, during or after conjugation, but preferably occurs before
conjugation. The effect of de-acetylation etc. can be assessed by
routine assays.
[0025] The Vi saccharide may be hydrolysed to form shortened
polysaccharides (e.g. with a degree of polymerisation (DP) of at
least 10, e.g. 20, 30, 40, 50, 60 or more) or oligosaccharides
(e.g. with a degree of polymerisation of from 2 to 10).
Oligosaccharides are preferred to polysaccharides for use in
vaccines. The average degree of polymerisation can conveniently be
measured by ion exchange chromatography or by colorimetric assays
[12].
[0026] In addition, it has been found by double immunodiffusion
that pectin, when O-acetylated at C-2 and C-3, is antigenically
identical to Vi. The structure of Vi differs from that of pectin in
that it is N-acetylated at C-2 and O-acetylated at C-3.
O-acetylated pectin conjugated to tetanus toxoid elicited Vi
antibodies in mice, and reinjection elicited a booster response
[13,14]. Accordingly, O-acetylated pectin may be used in the
invention in place of Vi. However, Vi conjugates have been shown to
be significantly more immunogenic than their O-acetylated pectin
analogs, and so Vi from natural sources is preferred [13].
Nevertheless, it will be understood that references to "Vi" may
include "O-acetylated pectin" and any other molecules that may be
structurally or antigenically identical to Vi and are capable of
eliciting antibodies that recognise native Vi.
[0027] "Vi" may refer to a Vi polysaccharide (e.g. with a degree of
polymerisation of at least 10, e.g. 20, 30, 40, 50, 60 or more) or
a Vi oligosaccharide (e.g. with a degree of polymerisation of from
2 to 10) and may have been chemically modified. Oligosaccharides
may be the result of depolymerisation and/or hydrolysis of a parent
polysaccharide.
Vi Purification
[0028] Capsular saccharides can be purified by known techniques, as
described in the references herein. A typical process involves base
extraction, centrifugation, filtration, RNase/DNase treatment,
protease treatment, concentration, size exclusion chromatography,
ultrafiltration, anion exchange chromatography, and further
ultrafiltration.
[0029] A particularly useful method is disclosed in reference 15,
which is incorporated herein by reference.
[0030] A process for purifying Vi may comprise the steps of (a)
precipitation of Vi, followed by (b) solubilisation of the
precipitated Vi using an alcohol, such as ethanol.
Precipitation and Alcohol Solubilisation
[0031] Many techniques for precipitating soluble polysaccharides,
such as Vi, are known in the art. Preferred methods use one or more
cationic detergents. The detergents preferably have the following
general formula:
##STR00001##
wherein: [0032] R.sub.1, R.sub.2 and R.sub.3 are the same or
different and each signifies alkyl or aryl; or R.sub.1 and R.sub.2
together with the nitrogen atom to which these are attached form a
5- or 6-membered saturated heterocyclic ring, and R.sub.3 signifies
alkyl or aryl; or R.sub.1, R.sub.2 and R.sub.3 together with the
nitrogen atom to which these are attached form a 5- or 6-membered
heterocyclic ring, unsaturated at the nitrogen atom, [0033] R.sub.4
signifies alkyl or aryl, and [0034] X.sup.- signifies an anion.
[0035] Particularly preferred detergents for use in the method are
tetrabutylammonium and cetyltrimethylammonium salts (e.g. the
bromide salts). Cetyltrimethylammonium bromide (`CTAB`) is
particularly preferred [16]. CTAB is also known as
hexadecyltrimethylammonium bromide, cetrimonium bromide, Cetavlon
and Centimide. Other detergents include hexadimethrine bromide and
myristyltrimethylammonium salts.
[0036] Vi can be released into media during culture. Accordingly,
the starting material for precipitation will typically be the
supernatant from a centrifuged bacterial culture or will be a
concentrated culture. This material may be filtered to remove
turbidity.
[0037] The precipitation step may be selective for Vi, but it will
typically also co-precipitate other components (e.g. proteins,
nucleic acid etc.).
[0038] Precipitated Vi may be collected by centrifugation prior to
solubilisation.
[0039] After precipitation, Vi (typically in the form of a complex
with the cationic detergent) is re-solubilised. It is preferred to
use a solvent which is relatively selective for Vi in order to
minimise contaminants (e.g. proteins, nucleic acid etc.). Ethanol
has been found to be advantageous in this respect, and it is highly
selective for the CTAB-Vi complex. Other lower alcohols may be used
(e.g. methanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol,
2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols etc.)
[0040] The alcohol is preferably added to the precipitated Vi to
give a final alcohol concentration (based on total content of
alcohol and water) of between 50% and 99% (e.g. around 55%, 60%,
65%, 70%, 75%, 80%, 85%, or around 90%), and preferably between 75%
and 95%.
[0041] The alcohol may be added to the precipitated Vi in pure form
or may be added in a form diluted with a miscible solvent (e.g.
water). Preferred solvent mixtures are alcohol:water mixtures, with
a preferred ratio of between around 70:30 and around 95:5 (e.g.
75:25, 80:20, 85:15, 90:10).
[0042] Compared with conventional processes for preparing capsular
polysaccharides, the two-step process of precipitation followed by
alcohol extraction is quicker and simpler.
[0043] In contrast to the process described in ref. 17, the process
uses cationic detergent rather than anionic detergent. Unlike the
process of ref. 18, precipitation does not require an inert porous
support.
[0044] Furthermore, unlike prior art processes, an alcohol is used
to re-solubilise Vi rather than to precipitate it.
Further Processing of the Solubilised Polysaccharide
[0045] After re-solubilisation, Vi is further treated to remove
contaminants because, in human vaccine production, even minor
contamination is not acceptable.
[0046] This treatment may include centrifugation of the solubilised
CTAB-Vi complex, followed by precipitation of Vi from the obtained
supernatant by exchanging cations (e.g. by the addition of calcium
or sodium salts) to give a Vi precipitate that is insoluble in
alcohol but soluble in water.
[0047] This precipitate may be collected by centrifugation and
further washed in alcohol and re-solubilised in an aqueous
solution, if desired.
[0048] The treatment process will also typically involve one or
more steps of filtration.
[0049] Depth filtration may be used. This is particularly useful
for clarification.
[0050] Filtration through activated carbon may be used. This is
useful for removing pigments and trace organic compounds. It can be
repeated until, for example, OD.sub.275 nm<0.2.
[0051] Size filtration or ultrafiltration may be used.
[0052] If Vi is hydrolysed, the hydrolysate will generally be sized
in order to remove short-length oligosaccharides. This can be
achieved in various ways, such as ultrafiltration followed by
ion-exchange chromatography.
[0053] The invention is not limited to saccharides purified from
natural sources, however, and the saccharides may be obtained by
other methods, such as total or partial synthesis.
Conjugates
[0054] Pure Vi is a poor immunogen. For protective efficacy,
therefore, Vi may be presented to the immune system as a Vi-carrier
conjugate. The use of conjugation to carrier proteins in order to
enhance the immunogenicity of carbohydrate antigens is well known
[e.g. reviewed in refs. 19 to 27 etc.] and is used in particular
for paediatric vaccines [28]. As described above, a saccharide may
be conjugated to a carrier protein or to a mixture of different
carrier proteins. Similarly, a carrier protein may carry a
saccharide or a mixture of different saccharides, i.e. multiple
different saccharides [29].
[0055] The invention provides a conjugate of (i) Vi, and (ii)
CRM.sub.197 as a carrier protein.
[0056] The CRM.sub.197 may be covalently conjugated to Vi directly
or via a linker.
[0057] Any suitable conjugation reaction can be used, with any
suitable linker where necessary. Oligosaccharides will typically be
sized prior to conjugation. Where the composition of the invention
includes a depolymerised saccharide, it is preferred that
depolymerisation precedes conjugation.
[0058] Attachment of Vi to CRM.sub.197 is preferably via a
--NH.sub.2 group e.g. in the side chain of a lysine residue in
CRM.sub.197, or of an arginine residue. Attachment to CRM.sub.197
may also be via a --SH group e.g. in the side chain of a cysteine
residue. Alternatively, Vi may be attached to CRM.sub.197 via a
linker molecule as described below.
[0059] Vi will typically be activated or functionalised prior to
conjugation. A preferred technique uses carbodiimides (e.g.
1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC)). Other
suitable techniques use hydrazides, active esters, norborane,
p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDAC, TSTU (see
also the introduction to reference 30).
[0060] Linkages via a linker group to carrier proteins in general
may be made using any known procedure, for example, the procedures
described in references 31 and 32. A useful type of linkage is an
adipic acid linker, which may be formed by coupling a free
--NH.sub.2 group (e.g. introduced to Vi by amination) with adipic
acid (using, for example, diimide activation), and then coupling a
protein to the resulting saccharide-adipic acid intermediate [23,
33, 34]. Another useful type of linkage is a carbonyl linker, which
may be formed by reaction of a free hydroxyl group of a modified Vi
with CDI [35, 36] followed by reaction with a protein to form a
carbamate linkage. A useful linker is adipic acid dihydrazide ADH
[37]. The carrier protein may be derivatised with ADH (for example,
by carbodiimide coupling at a carboxylic acid side group) and
subsequently attached to Vi [3] (again, for example, by
carbodiimide coupling). Other linkers include .beta.-propionamido
[38], nitrophenyl-ethylamine [39], haloacyl halides [40],
glycosidic linkages [41], 6-aminocaproic acid [42],
N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [43], C.sub.4
to C.sub.12 moieties [44], etc. Carbodiimide condensation can also
be used [45].
[0061] A useful process for linking Vi to CRM.sub.197 involves the
thiolation of Vi with cystamine or cysteamine (carbodiimide
coupling) and subsequent reaction with CRM.sub.197 derivatised with
SPDP [46].
[0062] Another useful process involves the introduction of amino
groups into Vi followed by derivatisation with an adipic diester
(e.g. adipic acid N-hydroxysuccinimido diester) and reaction with
CRM.sub.197.
[0063] A bifunctional linker may be used to provide a first group
for coupling to an amine group that has been introduced into Vi and
a second group for coupling to the carrier (typically for coupling
to an amine in the carrier).
[0064] The first group in the bifunctional linker is thus able to
react with an amine group (--NH.sub.2) on Vi. This reaction will
typically involve an electrophilic substitution of the amine's
hydrogen. The second group in the bifunctional linker is able to
react with an amine group on the carrier. This reaction will again
typically involve an electrophilic substitution of the amine.
[0065] Where the reactions with both Vi and the carrier protein
involve amines then it is preferred to use a bifunctional linker,
for example a homobifunctional linker of the formula X-L-X, where:
the two X groups are the same as each other and can react with the
amines; and where L is a linking moiety in the linker. A useful X
group is N-oxysuccinimide. L may have formula L'-L.sup.2-L', where
L' is carbonyl. Useful L.sup.2 groups are straight chain alkyls
with 1 to 10 carbon atoms (e.g. C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.1-10) e.g.
--(CH.sub.2).sub.4--.
[0066] Other X groups are those which form esters when combined
with HO-L-OH, such as norborane, p-nitrobenzoic acid, and
sulfo-N-hydroxysuccinimide.
[0067] Further bifunctional linkers for use with the invention
include acryloyl halides (e.g. chloride) and haloacylhalides.
[0068] The linker will generally be added in molar excess to
modified Vi.
[0069] Preferred carrier proteins are bacterial toxins; such as
diphtheria or tetanus toxins, or toxoids or mutants thereof. These
are commonly used in conjugate vaccines. The CRM.sub.197 diphtheria
toxin mutant is particularly preferred [47].
[0070] Other suitable carrier proteins include the N. meningitidis
outer membrane protein complex [48], synthetic peptides [49,50],
heat shock proteins [51,52], pertussis proteins [53,54], cytokines
[55], lymphokines [55], hormones [55], growth factors [55],
artificial proteins comprising multiple human CD4.sup.+ T cell
epitopes from various pathogen-derived antigens [56] such as N19
[57], protein D from H. influenzae [58-60], pneumolysin [61] or its
non-toxic derivatives [62], pneumococcal surface protein PspA [63],
iron-uptake proteins [64], toxin A or B from C. difficile [65],
recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [66], etc.
It is possible to use mixtures of carrier proteins. A single
carrier protein may carry multiple Vi saccharides [67].
[0071] Conjugates may have excess carrier protein (w/w) or excess
Vi (w/w) e.g. in the ratio range of 1:5 to 5:1. Conjugates with
excess carrier protein are typical e.g. in the range 0.2:1 to
0.9:1, such as 0.5:1, or with equal weights (1:1). In some
embodiments the Vi:protein ratio is between 0.4:1 and 1.2:1.
[0072] When the conjugate forms the Vi component in an immunogenic
composition of the invention, the composition may also comprise
free carrier protein [68].
[0073] The Vi moiety in the conjugate is preferably a low molecular
weight Vi polysaccharide or an oligosaccharide, as defined above.
Oligosaccharides will typically be sized prior to conjugation.
[0074] The protein-Vi conjugate is preferably soluble in water
and/or in a physiological buffer.
Pharmaceutical Compositions
[0075] The invention provides a pharmaceutical composition
comprising (a) Vi conjugate, and (b) a pharmaceutically acceptable
carrier. A thorough discussion of such carriers is available in
ref. 69.
[0076] Microbial infections affect various areas of the body and so
the compositions of the invention may be prepared in various forms.
For example, the compositions may be prepared as injectables,
either as liquid solutions or suspensions. Solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The composition may be prepared for topical
administration e.g. as an ointment, cream or powder. The
composition be prepared for oral administration e.g. as a tablet or
capsule, or as a syrup (optionally flavoured). The composition may
be prepared for pulmonary administration e.g. as an inhaler, using
a fine powder or a spray. The composition may be prepared as a
suppository or pessary. The composition may be prepared for nasal,
aural or ocular administration e.g. as drops, as a spray, or as a
powder [e.g. 70]. The composition may be included in a mouthwash.
The composition may be lyophilised.
[0077] The pharmaceutical composition is preferably sterile. It is
preferably pyrogen-free. It is preferably buffered e.g. at between
pH 6 and pH 8, generally around pH 7.
[0078] A composition of the invention may comprise a Vi conjugate
and saline.
[0079] The invention also provides a delivery device containing a
pharmaceutical composition of the invention. The device may be, for
example, a syringe or an inhaler.
[0080] Pharmaceutical compositions of the invention are preferably
immunogenic compositions, in that they comprise an immunologically
effective amount of Vi immunogen. By `immunologically effective
amount`, it is meant that the administration of that amount to an
individual, either in a single dose or as part of a series, is
effective for treatment or prevention. This amount varies depending
upon the health and physical condition of the individual to be
treated, age, the taxonomic group of individual to be treated (e.g.
non-human primate, primate, etc.), the capacity of the individual's
immune system to synthesise antibodies, the degree of protection
desired, the formulation of the vaccine, the treating doctor's
assessment of the medical situation, and other relevant factors. It
is expected that the amount will fall in a relatively broad range
that can be determined through routine trials. A dose of between 1
.mu.g and 20 .mu.g of saccharide is expected e.g. about 5
.mu.g/dose. Dosage treatment may be a single dose schedule or a
multiple dose schedule (e.g. including booster doses). The
composition may be administered in conjunction with other
immunoregulatory agents.
[0081] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals; in particular, human subjects can be treated.
[0082] Immunogenic compositions of the invention may be used
therapeutically (i.e. to treat an existing infection) or
prophylactically (i.e. to prevent future infection).
[0083] An immunogenic composition may be unadjuvanted. In other
embodiments, though, an immunogenic composition may include an
adjuvant, which can function to enhance the immune responses
(humoral and/or cellular) elicited in a patient who receives the
composition. Adjuvants that can be used with the invention include,
but are not limited to: [0084] A mineral-containing composition,
including calcium salts and aluminum salts (or mixtures thereof).
Calcium salts include calcium phosphate (e.g. the "CAP" particles
disclosed in ref. 71). Aluminum salts include hydroxides,
phosphates, sulfates, etc., with the salts taking any suitable form
(e.g. gel, crystalline, amorphous, etc.). Adsorption to these salts
is preferred. The mineral containing compositions may also be
formulated as a particle of metal salt [72]. The adjuvants known as
aluminum hydroxide and aluminum phosphate may be used. These names
are conventional, but are used for convenience only, as neither is
a precise description of the actual chemical compound which is
present (e.g. see chapter 9 of reference 155). The invention can
use any of the "hydroxide" or "phosphate" adjuvants that are in
general use as adjuvants. The adjuvants known as "aluminium
hydroxide" are typically aluminium oxyhydroxide salts, which are
usually at least partially crystalline. The adjuvants known as
"aluminium phosphate" are typically aluminium hydroxyphosphates,
often also containing a small amount of sulfate (i.e. aluminium
hydroxyphosphate sulfate). They may be obtained by precipitation,
and the reaction conditions and concentrations during precipitation
influence the degree of substitution of phosphate for hydroxyl in
the salt. The invention can use a mixture of both an aluminium
hydroxide and an aluminium phosphate. In this case there may be
more aluminium phosphate than hydroxide e.g. a weight ratio of at
least 2:1 e.g. .gtoreq.5:1, .gtoreq.6:1, .gtoreq.7:1, .gtoreq.8:1,
.gtoreq.9:1, etc. The concentration of Al.sup.+++ in a composition
for administration to a patient is preferably less than 10 mg/ml
e.g. .ltoreq.5 mg/ml, .ltoreq.4 mg/ml, .ltoreq.3 mg/ml, .ltoreq.2
mg/ml, .ltoreq.1 mg/ml, etc. A preferred range is between 0.3 and 1
mg/ml. A maximum of 0.85 mg/dose is preferred. [0085] Saponins
[chapter 22 of ref. 155], which are a heterologous group of sterol
glycosides and triterpenoid glycosides that are found in the bark,
leaves, stems, roots and even flowers of a wide range of plant
species. Saponin from the bark of the Quillaia saponaria Molina
tree have been widely studied as adjuvants. Saponin can also be
commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla
paniculata (brides veil), and Saponaria officianalis (soap root).
Saponin adjuvant formulations include purified formulations, such
as QS21, as well as lipid formulations, such as ISCOMs. QS21 is
marketed as Stimulon.TM.. Saponin compositions have been purified
using HPLC and RP-HPLC. Specific purified fractions using these
techniques have been identified, including QS7, QS17, QS18, QS21,
QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of
production of QS21 is disclosed in ref. 73. Saponin formulations
may also comprise a sterol, such as cholesterol [74]. Combinations
of saponins and cholesterols can be used to form unique particles
called immunostimulating complexs (ISCOMs) [chapter 23 of ref.
155]. ISCOMs typically also include a phospholipid such as
phosphatidylethanolamine or phosphatidylcholine. Any known saponin
can be used in ISCOMs. Preferably, the ISCOM includes one or more
of QuilA, QHA & QHC. ISCOMs are further described in refs.
74-76. Optionally, the ISCOMS may be devoid of additional detergent
[77]. A review of the development of saponin based adjuvants can be
found in refs. 78 & 79. [0086] Bacterial ADP-ribosylating
toxins (e.g. the E. coli heat labile enterotoxin "LT", cholera
toxin "CT", or pertussis toxin "PT") and detoxified derivatives
thereof, such as the mutant toxins known as LT-K63 and LT-R72 [80].
The use of detoxified ADP-ribosylating toxins as mucosal adjuvants
is described in ref. 81 and as parenteral adjuvants in ref. 82.
[0087] Bioadhesives and mucoadhesives, such as esterified
hyaluronic acid microspheres [83] or chitosan and its derivatives
[84]. [0088] Microparticles (i.e. a particle of .about.100 nm to
.about.150 nm in diameter, more preferably .about.200 nm to
.about.30 .mu.m in diameter, or .about.500 nm to .about.10 .mu.m in
diameter) formed from materials that are biodegradable and
non-toxic (e.g. a poly(.alpha.-hydroxy acid), a polyhydroxybutyric
acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.),
with poly(lactide-co-glycolide) being preferred, optionally treated
to have a negatively-charged surface (e.g. with SDS) or a
positively-charged surface (e.g. with a cationic detergent, such as
CTAB). [0089] Liposomes (Chapters 13 & 14 of ref. 155).
Examples of liposome formulations suitable for use as adjuvants are
described in refs. 85-87. [0090] Muramyl peptides, such as
N-acetylmuramyl-L-threonyl-D-isoglutamine ("thr-MDP"),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy
propylamide ("DTP-DPP", or "Theramide.TM.),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'
dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
("MTP-PE"). [0091] A polyoxidonium polymer [88,89] or other
N-oxidized polyethylene-piperazine derivative. [0092] A CD1d
ligand, such as an .alpha.-glycosylceramide [90-97] (e.g.
.alpha.-galactosylceramide), phytosphingosine-containing
.alpha.-glycosylceramides, OCH, KRN7000
[(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,-
4-octadecanetriol], CRONY-101, 3''-O-sulfo-galactosylceramide, etc.
[0093] A gamma inulin [98] or derivative thereof, such as
algammulin. [0094] An oil-in-water emulsion. Various such emulsions
are known, and they typically include at least one oil and at least
one surfactant, with the oil(s) and surfactant(s) being
biodegradable (metabolisable) and biocompatible. The oil droplets
in the emulsion are generally less than 5 .mu.m in diameter, and
may even have a sub-micron diameter, with these small sizes being
achieved with a microfluidiser to provide stable emulsions.
Droplets with a size less than 220 nm are preferred as they can be
subjected to filter sterilization. [0095] An immunostimulatory
oligonucleotide, such as one containing a CpG motif (a dinucleotide
sequence containing an unmethylated cytosine residue linked by a
phosphate bond to a guanosine residue), or a CpI motif (a
dinucleotide sequence containing cytosine linked to inosine), or a
double-stranded RNA, or an oligonucleotide containing a palindromic
sequence, or an oligonucleotide containing a poly(dG) sequence.
Immunostimulatory oligonucleotides can include nucleotide
modifications/analogs such as phosphorothioate modifications and
can be double-stranded or (except for RNA) single-stranded.
References 99, 100 and 101 disclose possible analog substitutions
e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine. The
adjuvant effect of CpG oligonucleotides is further discussed in
refs. 102-107. A CpG sequence may be directed to TLR9, such as the
motif GTCGTT or TTCGTI [108]. The CpG sequence may be specific for
inducing a Th1 immune response, such as a CpG-A ODN
(oligodeoxynucleotide), or it may be more specific for inducing a B
cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed
in refs. 109-111. Preferably, the CpG is a CpG-A ODN. Preferably,
the CpG oligonucleotide is constructed so that the 5' end is
accessible for receptor recognition. Optionally, two CpG
oligonucleotide sequences may be attached at their 3' ends to form
"immunomers". See, for example, references 108 & 112-114. A
useful CpG adjuvant is CpG7909, also known as ProMune.TM. (Coley
Pharmaceutical Group, Inc.). Another is CpG1826. As an alternative,
or in addition, to using CpG sequences, TpG sequences can be used
[115], and these oligonucleotides may be free from unmethylated CpG
motifs. The immunostimulatory oligonucleotide may be
pyrimidine-rich. For example, it may comprise more than one
consecutive thymidine nucleotide (e.g. TTTT, as disclosed in ref.
115), and/or it may have a nucleotide composition with >25%
thymidine (e.g. >35%, >40%, >50%, >60%, >80%, etc.).
For example, it may comprise more than one consecutive cytosine
nucleotide (e.g. CCCC, as disclosed in ref. 115), and/or it may
have a nucleotide composition with >25% cytosine (e.g. >35%,
>40%, >50%, >60%, >80%, etc.). These oligonucleotides
may be free from unmethylated CpG motifs. Immunostimulatory
oligonucleotides will typically comprise at least 20 nucleotides.
They may comprise fewer than 100 nucleotides.
[0096] A particularly useful adjuvant based around
immunostimulatory oligonucleotides is known as IC31.TM. [116]. Thus
an adjuvant used with the invention may comprise a mixture of (i)
an oligonucleotide (e.g. between 15-40 nucleotides) including at
least one (and preferably multiple) CpI motifs, and (ii) a
polycationic polymer, such as an oligopeptide (e.g. between 5-20
amino acids) including at least one (and preferably multiple)
Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a
deoxynucleotide comprising 26-mer sequence 5'-(IC).sub.13-3'. The
polycationic polymer may be a peptide comprising 11-mer amino acid
Lys-Leu-Lys-Leu.sub.5-Lys-Leu-Lys. [0097] 3-O-deacylated
monophosphoryl lipid A (`3dMPL`, also known as `MPL.TM.`)
[117-120]. In aqueous conditions, 3dMPL can form micellar
aggregates or particles with different sizes e.g. with a diameter
<150 nm or >500 nm. Either or both of these can be used with
the invention, and the better particles can be selected by routine
assay. Smaller particles (e.g. small enough to give a clear aqueous
suspension of 3dMPL) are preferred for use according to the
invention because of their superior activity [121]. Preferred
particles have a mean diameter less than 220 nm, more preferably
less than 200 nm or less than 150 nm or less than 120 nm, and can
even have a mean diameter less than 100 nm. In most cases, however,
the mean diameter will not be lower than 50 nm. [0098] Methyl
inosine 5'-monophosphate ("MIMP") [122]. [0099] A polyhydroxlated
pyrrolizidine compound [123], such as one having formula:
[0099] ##STR00002## [0100] where R is selected from the group
comprising hydrogen, straight or branched, unsubstituted or
substituted, saturated or unsaturated acyl, alkyl (e.g.
cycloalkyl), alkenyl, alkynyl and aryl groups, or a
pharmaceutically acceptable salt or derivative thereof. Examples
include, but are not limited to: casuarine,
casuarine-6-.alpha.-D-glucopyranose, 3-epi-casuarine,
7-epi-casuarine, 3,7-diepi-casuarine, etc. [0101] An
imidazoquinoline compound, such as Imiquimod ("R-837") [124,125],
Resiquimod ("R-848") [126], and their analogs; and salts thereof
(e.g. the hydrochloride salts). Further details about
immunostimulatory imidazoquinolines can be found in references 127
to 131. [0102] A thiosemicarbazone compound, such as those
disclosed in reference 132. Methods of formulating, manufacturing,
and screening for active compounds are also described in reference
132. The thiosemicarbazones are particularly effective in the
stimulation of human peripheral blood mononuclear cells for the
production of cytokines, such as TNF-.alpha.. [0103] A tryptanthrin
compound, such as those disclosed in reference 133. Methods of
formulating, manufacturing, and screening for active compounds are
also described in reference 133. The thiosemicarbazones are
particularly effective in the stimulation of human peripheral blood
mononuclear cells for the production of cytokines, such as
TNF-.alpha.. [0104] A nucleoside analog, such as: (a) Isatorabine
(ANA-245; 7-thia-8-oxoguanosine):
[0104] ##STR00003## [0105] and prodrugs thereof; (b) ANA975; (c)
ANA-025-1; (d) ANA380; (e) the compounds disclosed in references
134 to 136Loxoribine (7-allyl-8-oxoguanosine) [137]. [0106]
Compounds disclosed in reference 138, including: Acylpiperazine
compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ)
compounds, Benzocyclodione compounds, Aminoazavinyl compounds,
Aminobenzimidazole quinolinone (ABIQ) compounds [139,140],
Hydrapthalamide compounds, Benzophenone compounds, Isoxazole
compounds, Sterol compounds, Quinazilinone compounds, Pyrrole
compounds [141], Anthraquinone compounds, Quinoxaline compounds,
Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole
compounds [142]. [0107] An aminoalkyl glucosaminide phosphate
derivative, such as RC-529 [143,144]. [0108] A phosphazene, such as
poly[di(carboxylatophenoxy)phosphazene] ("PCPP") as described, for
example, in references 145 and 146. [0109] A compound of formula I,
II or III, or a salt thereof:
[0109] ##STR00004## [0110] as defined in reference 147, such as `ER
803058`, `ER 803732`, `ER 804053`, ER 804058', `ER 804059`, `ER
804442`, `ER 804680`, `ER 804764`, ER 803022 or `ER 804057`
e.g.:
[0110] ##STR00005## [0111] Derivatives of lipid A from Escherichia
coli such as OM-174 (described in refs. 148 & 149). [0112]
Compounds containing lipids linked to a phosphate-containing
acyclic backbone, such as the TLR4 antagonist E5564 [150,151]:
##STR00006##
[0113] These and other adjuvant-active substances are discussed in
more detail in references 155 & 156.
[0114] Antigens and adjuvants in a composition will typically be in
admixture.
[0115] Compositions may include two or more of said adjuvants. For
example, they may advantageously include both an oil-in-water
emulsion and 3dMPL, etc.
[0116] Specific oil-in-water emulsion adjuvants useful with the
invention include, but are not limited to: [0117] A submicron
emulsion of squalene, Tween 80, and Span 85. The composition of the
emulsion by volume can be about 5% squalene, about 0.5% polysorbate
80 and about 0.5% Span 85. In weight terms, these ratios become
4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant
is known as `MF59` [152-154], as described in more detail in
Chapter 10 of ref. 155 and chapter 12 of ref. 156. The MF59
emulsion advantageously includes citrate ions e.g. 10 mM sodium
citrate buffer. [0118] An emulsion of squalene, a tocopherol, and
Tween 80. The emulsion may include phosphate buffered saline. It
may also include Span 85 (e.g. at 1%) and/or lecithin. These
emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol
and from 0.3 to 3% Tween 80, and the weight ratio of
squalene:tocopherol is preferably .ltoreq.1 as this provides a more
stable emulsion. Squalene and Tween 80 may be present volume ratio
of about 5:2. One such emulsion can be made by dissolving Tween 80
in PBS to give a 2% solution, then mixing 90 ml of this solution
with a mixture of (5 g of DL-.alpha.-tocopherol and 5 ml squalene),
then microfluidising the mixture. The resulting emulsion may have
submicron oil droplets e.g. with an average diameter of between 100
and 250 nm, preferably about 180 nm. [0119] An emulsion of
squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100).
The emulsion may also include a 3d-MPL (see below). The emulsion
may contain a phosphate buffer. [0120] An emulsion comprising a
polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton
X-100) and a tocopherol (e.g. an .alpha.-tocopherol succinate). The
emulsion may include these three components at a mass ratio of
about 75:11:10 (e.g. 750 .mu.g/ml polysorbate 80, 110 .mu.g/ml
Triton X-100 and 100 .mu.g/ml .alpha.-tocopherol succinate), and
these concentrations should include any contribution of these
components from antigens. The emulsion may also include squalene.
The emulsion may also include a 3d-MPL (see below). The aqueous
phase may contain a phosphate buffer. [0121] An emulsion of
squalane, polysorbate 80 and poloxamer 401 ("Pluronic.TM. L121").
The emulsion can be formulated in phosphate buffered saline, pH
7.4. This emulsion is a useful delivery vehicle for muramyl
dipeptides, and has been used with threonyl-MDP in the "SAF-1"
adjuvant [157] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121
and 0.2% polysorbate 80). It can also be used without the Thr-MDP,
as in the "AF" adjuvant [158] (5% squalane, 1.25% Pluronic L121 and
0.2% polysorbate 80). Microfluidisation is preferred. [0122] An
emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid,
and 0.05-5% of a non-ionic surfactant. As described in reference
159, preferred phospholipid components are phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidylglycerol, phosphatidic acid, sphingomyelin and
cardiolipin. Submicron droplet sizes are advantageous. [0123] A
submicron oil-in-water emulsion of a non-metabolisable oil (such as
light mineral oil) and at least one surfactant (such as lecithin,
Tween 80 or Span 80). Additives may be included, such as QuilA
saponin, cholesterol, a saponin-lipophile conjugate (such as
GPI-0100, described in reference 160, produced by addition of
aliphatic amine to desacylsaponin via the carboxyl group of
glucuronic acid), dimethyldioctadecylammonium bromide and/or
N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine. [0124] An
emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g.
a cholesterol) are associated as helical micelles [161].
Medical Treatments and Uses
[0125] The invention also provides a Vi conjugate of the invention,
for use in medicine e.g. for use in raising an antibody response in
a mammal.
[0126] The invention also provides a method for raising an immune
response in a mammal, comprising administering a Vi conjugate or
pharmaceutical composition of the invention to the mammal.
[0127] The invention also provides the use of a Vi conjugate of the
invention in the manufacture of a medicament for preventing or
treating typhoid fever in a mammal.
[0128] The immune response raised by these methods and uses will
generally include an antibody response, preferably a protective
antibody response. Methods for assessing antibody responses after
saccharide immunisation are well known in the art. The antibody
response is preferably an IgA or IgG response. The immune response
may be prophylactic and/or therapeutic. The mammal is preferably a
human.
[0129] Compositions of the invention will generally be administered
directly to a patient. Direct delivery may be accomplished by
parenteral injection (e.g. subcutaneously, intraperitoneally,
intravenously, intramuscularly, or to the interstitial space of a
tissue), or by rectal, oral, vaginal, topical, transdermal,
intradermal, ocular, nasal, aural, or pulmonary administration.
Injection or intranasal administration is preferred.
[0130] The invention may be used to elicit systemic and/or mucosal
immunity.
[0131] Vaccines prepared according to the invention may be used to
treat both children (including infants) and adults. Thus a subject
may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55
years old, or at least 55 years old. Preferred subjects for
receiving the vaccines are the young (e.g. .ltoreq.5 years old).
The vaccines are not suitable solely for these groups, however, and
may be used more generally in a population.
[0132] Treatment can be by a single dose schedule or a multiple
dose schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. In a multiple
dose schedule the various doses may be given by the same or
different routes e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc. Administration of more
than one dose (typically two doses) is particularly useful in
immunologically naive patients. Multiple doses will typically be
administered at least 1 week apart (e.g. about 2 weeks, about 3
weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks,
about 12 weeks, about 16 weeks, etc.). An example schedule provides
a first dose at 6 weeks of age and a second dose at 10 weeks of
age, to coincide with existing infant immunisations
(co-administration with EPI vaccines). This primary schedule may be
followed by a booster dose after a child's first birthday.
[0133] Conjugates of the invention may be combined with non-Vi
antigens into a single composition for simultaneous immunisation
against multiple pathogens. As an alternative to making a combined
vaccine, conjugates may be administered to patients at
substantially the same time as (e.g. during the same medical
consultation or visit to a healthcare professional or vaccination
centre) other vaccines. Antigens for use in these combination
vaccines or for concomitant administration include, for instance,
immunogens from Streptococcus agalactiae, Staphylococcus aureus
and/or Pseudomonas aeuruginosa, hepatitis A virus, hepatitis B
virus, Neisseria meningitidis (such as saccharides or conjugated
saccharides, for serogroups A, C, W135 and/or Y), Streptococcus
pneumoniae (such as saccharides or conjugated saccharides),
etc.
[0134] In one embodiment, a composition may comprise a Vi conjugate
of the invention in combination with a Salmonella paratyphi A
antigen, such as an H or O antigen (e.g. an 0:2 saccharide
antigen), to provide a bivalent typhoid vaccine. In another
embodiment, a composition may comprise a Vi conjugate of the
invention in combination with a Salmonella typhimurium antigen,
such as an H or O antigen (e.g. an 0:9 saccharide). In another
embodiment, a composition may comprise a Vi conjugate of the
invention in combination with a Salmonella enteritidis antigen,
such as an H or O antigen (e.g. an O:4,5 saccharide).
DEFINITIONS
[0135] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0136] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0137] The term "about" in relation to a numerical value x is
optional and means, for example, x+10%.
[0138] Where animal (and particularly bovine) materials are used in
the culture of cells, they should be obtained from sources that are
free from transmissible spongiform encaphalopathies (TSEs), and in
particular free from bovine spongiform encephalopathy (BSE).
Overall, it is preferred to culture cells in the total absence of
animal-derived materials.
[0139] Where a compound is administered to the body as part of a
composition then that compound may alternatively be replaced by a
suitable prodrug.
BRIEF DESCRIPTION OF DRAWINGS
[0140] FIG. 1 depicts the structural formula of S. typhi Vi
(.alpha.1,4-N-acetylgalactosaminouronic acid).
[0141] FIG. 2 shows a reaction scheme for the preparation of
derivatised proteins.
[0142] FIG. 3 shows a SEC analysis of CRM.sub.197 and CRM.sub.197
derivatised with ADH.
[0143] FIG. 4 shows a SEC analysis of tetanus toxoid derivatised
with ADH.
[0144] FIG. 5 shows SDS page patterns of, from left to right, TT,
TT.sub.ADH, CRM and CRM.sub.ADH.
[0145] FIG. 6 shows a reaction scheme for the preparation of a
Vi-protein conjugate.
[0146] FIG. 7 and FIG. 8 show gel filtration profiles of Vi and
tetanus toxoid on Sephacryl S-1000.
[0147] FIG. 9 shows a gel filtration profile of "pool 1", and FIG.
10 shows a profile of "pool 2".
[0148] FIG. 11 shows a SEC analysis of Vi-TT.sub.ADH.
[0149] FIG. 12 shows a SDS-PAGE profile (gel 3-8%) of
Vi-CRM.sub.ADH reaction mixtures after dialysis. Lane 2 is
CRM.sub.ADH (5 .mu.g), Lanes 3-5 are 10 .mu.l of the reactions
mixtures after dialysis of Lots 04-06 respectively.
[0150] FIG. 13 shows the purification of Lot 06 on Sephacryl
S-1000.
[0151] FIG. 14 shows a SDS-PAGE profile (gel 3-8%) of:
"2"-CRM.sub.ADH 3 .mu.g, "3"-reaction mixture 10 .mu.l and
fractions 1-11 and 12-22 of the purification of Lot 06.
[0152] FIGS. 15 to 17 show SEC analyses comparing pools 1 obtained
from Lots 04-06, pools 2 obtained from Lots 04-06, and pools 1 and
2 obtained from Lot 06.
[0153] FIG. 18 shows the average anti-Vi antibody values. FIGS. 18A
and 18B show data from different lots of conjugates, but the
control groups are the same in each. From left to right the groups
in both 18A and 18B are: PBS; Vi; Vi+CRM197; Vi+TT; Vi-CRM197;
Vi-TT; Vi-CRM197 with alum; Vi-TT with alum; and Vi-CRM197 with
CFA+IFA.
MODES FOR CARRYING OUT THE INVENTION
Vi Purification
[0154] The supernatant of a 5 L sample of C. freundii WR7001 in
mod-LB was concentrated (20 times) to 250 ml with a 100K membrane.
The sample was diafiltered against NaCl 1 M (2.5 L) and then with
water (1.5 L), again with a 100K membrane, and the permeate
discarded. A 0.22 .mu.m filter was used to remove turbidity.
Subsequently, 0.9% CTAB was added to form a Vi-CTAB precipitate.
This was centrifuged at 18000 g for 15 minutes and the supernatant
discarded.
[0155] The precipitate was suspended in ethanol (96%, 110 ml) and
mixed overnight at RT before subsequent centrifugation at 18000 g
for 50 minutes, after which the resulting precipitate was
discarded. 0.1 M NaCl was added to the supernatant to form a gel
which was centrifuged at 18000 g for 10 minutes. The precipitate
was collected and washed with ethanol, then solubilised in aqueous
NaCl (1 M, 50 ml) and filtered. The retentate was brought to 80%
ethanol and centrifuged at 18000 g for 10 minutes. The precipitate
was again washed with ethanol. One part of the precipitate remained
in suspension (Lot A). The two parts were collected separately as
Lot A (97 mg) and Lot B (170 mg).
[0156] In a modified process, the supernatant was concentrated to
8.0-10.0 g/L with a 100K membrane. The sample was diafiltered
against NaCl 1 M, Tris 0.1M, EDTA 0.02M pH 7.3 (2.5 L) and then
with water (2.5 L), again with a 100K membrane, and the permeate
was discarded. Subsequently, 2.0% CTAB was added to form a Vi-CTAB
precipitate. The precipitate was centrifuged at 18000 g for 15
minutes and the supernatant discarded. The precipitate was then
washed with water, centrifuged and resuspended in ethanol (85%) and
mixed until completely solubilised. The solution was passed through
SP10 and carbon filters. The filtrate was precipitated with NaCl
0.2 M and centrifuged at 18000 g for 5 minutes. The precipitate was
resuspended in NaCl 1.0 M to give a concentration of 3-5 mg/mL.
This solution was diafiltrated against water and 0.22 .mu.M
filtered.
[0157] The purification process has a good yield and provides
saccharide with good purity (less than 0.5% protein, less than
0.01% nucleic acid) and is gentle enough to preserve high levels of
0-acetylation.
Tetanus Toxoid Derivatisation
[0158] ADH (3.25 per mg of protein) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-hydrochloride (EDAC)
(EDAC/protein=0.152 w/w) were added to tetanus toxoid (in
2-(N-morpholino)ethanesulfonic acid (MES) buffer 50-100 mM, pH 6.18
to 6.20). The reaction was carried out for 1 hour at room
temperature.
[0159] The reaction mixture was dialysed against 0.2 M NaCl, 5 mM
MES buffer, pH 7.05, 2-8.degree. C. and 5 mM MES buffer, pH 7.00,
2-8.degree. C. Derivatised tetanus toxoid (86% yield) was
obtained.
CRM.sub.197 Derivatisation
[0160] ADH (3.5 mg per mg of protein) and EDAC (EDAC/protein=0.15
w/w) were added to CRM.sub.197 (11.5 mg/ml in MES buffer 50-100 mM,
pH 6.0). The reaction was carried out for 1 hour at room
temperature, pH 6.0-6.2. Maintenance of the pH in this range
prevented protein precipitation.
[0161] The reaction mixture was dialyzed overnight against 5 mM MES
buffer, NaCl 0.2 M, pH 7.0 and then against 5 mM MES buffer, pH 7.0
at 4.degree. C. Protein was measured by microBCA analysis (yield of
75-85%). Sucrose 10% w/v was added to the product and it was stored
at -20.degree. C. Rather than store with sucrose, though, it can be
stored in 5 mM MES, pH 7.0.
Derivatised Protein Characterisation
[0162] Derivatisation of the proteins with ADH was verified by a
colorimetric method (TNBS method).
[0163] The molar ratio of ADH to TT was measured by MS Maldi-T of
as about 11.
[0164] The molar ratio of ADH to CRM.sub.197 was measured by MS
Q-Tof. This showed the formation of several products characterised
by the presence of a different number of linkers bound to the
protein (from 3 to 10, the principal product containing 6 bound
linkers).
[0165] The derivatised proteins (and, subsequently, the conjugates)
were examined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) using 3-8% tris-acetate gels (NuPAGE).
The samples (5-20 .mu.l to have a protein content of 5 .mu.g) were
added of DTT 0.5M (1/5 v/v) and of NuPAGE LDS Sample Buffer (1/5
v/v). The mixtures were heated at 100.degree. C. for 1 minute and
the samples applied to the wells. The gel was subjected to
electophoresis at 30 mA in Tris-Acetate SDS Running Buffer
(Invitrogen). At the end it was stained with Simply Blue Safe Stain
(Invitrogen).
[0166] SDS-PAGE patterns and SEC profiles (214 nm, TSK gel 4000;
phosphate buffer 100 mM+NaCl 100 mM+5% CH.sub.3CN, pH 7.0) of the
derivatised proteins were found to be similar to those of the
native proteins (see FIG. 3 to FIG. 5).
Vi Conjugation to Derivatised Tetanus Toxoid
[0167] EDAC (4.4 mg) was added to Vi (4.6 mg) in a buffered
solution at pH 6.08 (1.5 ml 200 mM MES buffer) and allowed to react
for 2 minutes at room temperature.
[0168] Derivatised tetanus toxoid (9.2 mg) in a buffered solution
at pH 7.0 (1.5 ml 5 mM MES buffer) was allowed to react with the
activated Vi for 3 hours at room temperature ([TT.sub.ADH]=3.15 mg
ml.sup.-1, [Vi]=1.53 mg ml.sup.-1, Ratio TT.sub.ADH/Vi (w)=2,
[EDAC]=1.5 mg ml.sup.-1).
[0169] The reaction mixture was dialysed against 0.2 M NaCl, 10 mM
phosphate buffer, pH 7.07, 4.degree. C. and purified by Sephacryl
S-1000 (1.5.times.90 cm) in 10 mM phosphate buffer, 200 mM NaCl, pH
7.00. During conjugate purification, two different pools were
collected (see FIG. 7), which were characterised by a different MW.
Gel filtration profiles of Vi and of TT on Sephacryl S-1000 show
(see FIG. 7 and FIG. 8): [0170] conjugate purification from the
free protein is feasible (pool 1). [0171] conjugate purification
from free saccharide is not feasible (pool 2--free Vi co-elutes
with the conjugate).
[0172] Pool 1 should therefore contain less free Vi than pool
2.
[0173] FIG. 9 shows the profile on Sephacryl S1000 (1.6.times.90
cm) of the un-conjugated protein with 10 mM phosphate, NaCl 200 mM,
pH 7 at a flow of 0.2 ml/min. FIG. 10 shows the profile on
Sephacryl S1000 (in the same conditions) of free Vi.
[0174] Pools 1 and 2 were dialysed against 2 mM phosphate buffer pH
7.0 and their contents were as follows:
TABLE-US-00001 Protein Saccharide Ratio (w/w) Conjugate content
content saccharide/ Vi-TT.sub.ADH (micro BCA) (acridine orange)
protein Pool 1 2.46 mg 1.02 mg 0.41 Pool 2 5.56 mg 3.73 mg 0.67
[0175] The conjugate was characterised by SEC analysis (see FIG.
11, 214 nm, TSK gel 6000; phosphate buffer 100 mM+NaCl 100 mM+5%
CH.sub.3CN, pH 7.0): [0176] PSVi-TT.sub.ADH pool 1: polysaccharide
26.8 .mu.g/ml; protein 64.64 .mu.g/ml [0177] PSVi-TT.sub.ADH pool
2: polysaccharide 82.8 .mu.g/ml; protein 123.68 .mu.g/ml [0178]
PSVi: 1.4 mg/ml [0179] TT.sub.ADH: 0.1 mg/ml
Vi Conjugation to Derivatised CRM.sub.197
[0180] Three different Vi-CRM.sub.ADH Lots ("04", "05", and "06")
were prepared as follows:
[0181] EDAC (4.4 mg) was added to Vi (4.6 mg, giving a EDAC:Vi
molar ratio of about 1.43:1) in a buffered solution at pH 6.0 (1.65
ml 20 mM MES buffer) and mixed for 2 minutes at room temperature.
(In later experiments, for comparison, the amount of EDAC was
reduced, using molar ratios of 5:1 and 9:1. CRM197 conjugates
obtained with these derivatised saccharides were better
characterisable and reproducible with <5% of unconjugated
CRM-ADH. The ratio 5:1 was better than 1.4:1, and the ratio 9:1 was
better than 5:1).
[0182] Derivatised CRM.sub.197 (9.2 mg) in a buffered solution at
pH 7.0 (1.085 ml 5 mM MES buffer) was allowed to react with the
activated Vi for 3 hours at room temperature ([CRM.sub.ADH]=3.07 mg
ml.sup.-1, [Vi]=1.53 mg ml.sup.-1, Ratio CRM.sub.ADH/Vi(w)=2,
[EDAC]=1.47 mg ml.sup.-1), during which the pH was maintained at
6.0-6.20 by using MES buffer in order to avoid precipitation.
[0183] As noted above, CRM.sub.ADH is added in 5 mM MES buffer, pH
7. It is necessary that the final mixture is in MES buffer not
lower than 50-60 mM at pH 6 to maintain the pH constant during the
reaction itself.
[0184] The reaction mixture was purified by Sephacryl S1000 column
(1.5.times.90 cm) in 10 mM sodium phosphate buffer, 200 mM NaCl, pH
7 at 4.degree. C.
[0185] As an alternative means of purification, CRM.sub.ADH can be
removed from the conjugate by tangential ultrafiltration (100K or
300K membrane) as follows: reaction mixture diluted from 15 to 50
ml with 10 mM phosphate buffer pH 7.2; membrane: Vivaflow 200
cm.sup.2 100K (regenerated cellulose); P.sub.in: 1.2-1.3 bar;
P.sub.out: 0.4 bar; flow: 22.4 ml/min; permeate volume: 1.4 L; (28
cycles with 10 mM phosphate buffer pH 7.2); final retentate volume
of 152 ml.
[0186] The conjugate was characterised by microBCA (protein
content), acridine orange titration and NMR/HPAEC-PAD (saccharide
content), .sup.1H NMR (O-acetyl level and EDAC derivative
quantification), HPLC and SDS-PAGE.
[0187] FIG. 13 shows the purification of Lot 06 on Sephacryl S-1000
(Sephacryl S1000 1.6 cm.times.90 cm; Flow: 0.2 ml/min, Eluent: 200
mM NaCl, 10 mM NaH.sub.2PO.sub.4, pH 7.0). Fractions (1-11 and
12-22 for Lot 06) were collected for each Lot in two different
pools, distinguished by MW. The first pool (earlier fractions) was
purified of free saccharide, whilst the second contained an
undetermined amount of free saccharide.
[0188] The pools were dialyzed against 2 mM NaH.sub.2PO.sub.4, pH
7.5, at 4.degree. C., overnight and contents were:
TABLE-US-00002 Saccharide Saccharide Protein Ratio PS/protein (w/w)
conc. (.mu.g/ml) conc. (.mu.g/ml) conc. HPAEC-PAD or % yield in %
yield in saccharide Conjugate HPAEC-PAD acridine orange (.mu.g/ml)
acridine orange protein (HPAEC-PAD) Vi-CRM.sub.ADH 24.37 32.75
52.06 0.47 0.63 64.90 84.31 Lot04 Pool 1 Vi-CRM.sub.ADH 78.36 59.15
106.1 0.74 0.56 Lot04 Pool 2 Vi-CRM.sub.ADH 34.51 35.95 63.17 0.55
0.57 63.94 86.82 Lot05 Pool 1 Vi-CRM.sub.ADH 71.39 64.6 93.42 0.76
0.69 Lot05 Pool 2 Vi-CRM.sub.ADH 39.12 29.55 59.1 0.66 0.5 56.89
86.23 Lot06 Pool 1 Vi-CRM.sub.ADH 67.73 50 82.41 0.82 0.61 Lot06
Pool 2
[0189] FIGS. 15 to 17 show comparative SEC analyses of these pools
(TSKgel 6000PW (TosoHaas) analytical column (7.5 mm.times.30.0 cm),
eluent: 100 mM NaCl, 100 mM NaH.sub.2PO.sub.4, 5% CH.sub.3CN, pH
7.2; Flow: 0.5 mL/min; V.sub.0: .about.13.5 min-V.sub.t:
.about.27.8 min).
[0190] The conjugate was sterile filtered (0.22 .mu.m), aliquoted
and stored at -80.degree. C. (4.degree. C.).
Determination of In Vivo Activity
Antigens Used
[0191] The Vi-TT conjugates prepared in the previous example were
used. Vi-CRM.sub.ADH Lot 03 was prepared using substantially the
same conditions as Vi-CRM.sub.ADH Lots 04 to 06. Vi-CRM.sub.ADH Lot
01 was also prepared using substantially the same conditions as
Vi-CRM.sub.ADH Lots 04 to 06, but with the difference that the
ratio CRM.sub.ADH/Vi (w) was equal to 1. The hyperimmune mouse
serum immunized with Vi-rEPA was obtained from the NIH.
Conjugate Characteristics
TABLE-US-00003 [0192] Protein Saccharide Ratio (w/w) content
content saccharide/ Conjugate (micro BCA) (acridine orange) protein
a) Vi-CRM.sub.ADH Lot 01 3.18 mg 3.65 mg 1.15 b) Vi-CRM.sub.ADH Lot
03 4.13 mg 2.93 mg 0.71 Pool 2 c) Vi-TT.sub.ADH Pool 1 2.46 mg 1.02
mg 0.41 d) Vi-TT.sub.ADH Pool 2 5.56 mg 3.73 mg 0.67
Study Design
TABLE-US-00004 [0193] Dose N. in- Group Immunization (.mu.g)
jections Bleed days Route 1 PBS -- 3 0, 14, 28, 42 SC 2 Vi 2.5 3 0,
14, 28, 42 SC 3 Vi + CRM.sub.ADH 2.5 3 0, 14, 28, 42 SC 4 Vi +
TT.sub.ADH 2.5 3 0, 14, 28, 42 SC 5 Vi-CRM.sub.ADH Lot 01 2.5 3 0,
14, 28, 42 SC 6 Vi-CRM.sub.ADH Lot 03 2.5 3 0, 14, 28, 42 SC Pool 2
7 Vi-TT.sub.ADH Pool 1 2.5 3 0, 14, 28, 42 SC 8 Vi-TT.sub.ADH Pool
2 2.5 3 0, 14, 28, 42 SC 9.sup.a Vi-CRM.sub.ADH Lot 01 2.5 3 0, 14,
28, 42 SC 10.sup.a Vi-CRM.sub.ADH Lot 03 2.5 3 0, 14, 28, 42 SC
Pool 2 11.sup.a Vi-TT.sub.ADH Pool 1 2.5 3 0, 14, 28, 42 SC
12.sup.a Vi-TT.sub.ADH Pool 2 2.5 3 0, 14, 28, 42 SC 13.sup.b
Vi-CRM.sub.ADH Lot 03 10 1-2-3 0, 14, 28, 42 SC Pool 2 14.sup.b
Vi-CRM.sub.ADH Lot 03 10 1-2-3 0, 14, 28, 42 SC Pool 2
.sup.aconjugates were adjuvanted with alum .sup.b1.sup.st
immunization was adjuvanted with CFA, 2.sup.nd and 3.sup.rd
immunizations with IFA
Immunizations
[0194] Balb/c female mice were divided in fourteen groups of eight
mice each and were subcutaneously immunized with 2.5 .mu.g of Vi,
Vi-conjugate, or a physical mixture of Vi and ADH-derivatized
carrier protein. Only groups 13 and 14 received 10 .mu.g of
immunization dose.
[0195] Three injections of 200 .mu.l each were given every two
weeks, with bleedings two weeks after each immunization.
[0196] Groups 9 to 12 received alum as adjuvant, while the adjuvant
for groups 13 & 14 was complete Freund's adjuvant (CFA, 1st
injection) and incomplete Freund's adjuvant (IFA, 2nd & 3rd
injection).
ELISA Method
[0197] The wells of 96-well ELISA plates (Maxisorp, Nunc) were
coated with 100 .mu.l of 1 .mu.g/ml Vi in carbonate buffer (0.05M,
pH: 9.6) and left overnight at 4.degree. C. Vi used for coating was
purified from Citrobacter freundii WR7011. The following morning,
the plates were blocked with 200 .mu.l/well of 5% fat-free milk in
PBS-Tween 20 (PBST, 0.05%) for 1 hour at room temperature (RT).
After washing with PBST, 100 .mu.l/well of mouse sera (1:200
diluted in PBST with 0.1% BSA) were incubated for 2 hours at RT.
After three more washes, alkaline phosphatase-conjugated goat
anti-mouse IgG secondary antibody (Sigma A3438, 1: 10000 diluted in
PBST, 0.1% BSA) was incubated at 100 .mu.l/well for 1 hour at RT.
Alkaline phosphatase substrate (p-NPP, Sigma N2765) was solved in
diethanolamine buffer (1M, pH: 9.8) and was incubated after another
wash for 1 hour at RT. Plates were read at 405 and 490 nm using an
ELISA reader. Absorbance values used for antibody units
determination were obtained by subtracting 490 nm to 405 nm values.
Antibody units are expressed relative to a hyperimmune mouse
anti-Vi standard serum, after Hill Plot analysis.
[0198] A hyperimmune mouse serum immunized with Vi-rEPA was used as
internal positive control.
[0199] Each mouse serum was run in triplicate on three different
ELISA plates and data are presented as arithmetic means and
standard errors. One antibody unit is defined as the reciprocal of
the dilution of the standard sera that gives an optical density
equal to 1 in a standard ELISA.
Anti-CRM Antibody Values in Groups 1, 2, 3, 5, 6, 9, 10, 13, 14
TABLE-US-00005 [0200] Average Standard Error Group Immunization T14
T28 T42 T14 T28 T42 1 PBS -0.41 -0.64 -0.77 0.12 0.14 0.09 2 Vi
-0.89 -0.58 -0.65 0.14 0.17 0.07 3 Vi + CRM 0.09 34.88 256.81 0.25
16.11 42.99 5 Vi-CRM.sub.ADH Lot 01 3.80 149.80 311.68 1.19 47.20
49.33 6 Vi-CRM.sub.ADH Lot 03 Pool 2 7.30 163.06 550.38 2.64 50.91
44.70 9 Vi-CRM.sub.ADH Lot 01/alum 14.86 379.57 721.00 5.98 49.88
34.96 10 Vi-CRM.sub.ADH Lot 03 Pool 2/alum 44.73 263.46 452.91 7.27
16.74 29.09 13 Vi-CRM.sub.ADH Lot 01 50.91 586.61 762.92 15.29
41.35 65.50 14 Vi-CRM.sub.ADH Lot 01 69.18 672.56 841.66 22.68
53.35 43.42
Anti-Vi Antibody Values in all Groups
TABLE-US-00006 [0201] Average Standard Error Group N. Immunization
T14 T28 T42 T14 T28 T42 1 PBS 3.3 -1.03 -1.54 0.5 0.81 0.52 2 Vi
-3.4 -1.94 -0.04 0.4 0.52 0.52 3 Vi + CRM 5.0 -0.14 0.03 0.8 1.04
0.65 4 Vi + TT 1.1 2.51 1.31 1.3 1.23 0.39 5 Vi-CRM.sub.ADH Lot 01
53.7 242.20 191.53 8.4 31.40 40.50 6 Vi-CRM.sub.ADH Lot 03 Pool 2
95.4 245.39 225.66 40.9 45.33 38.00 7 Vi-TT.sub.ADH Pool 1 75.5
170.20 160.49 18.0 27.17 34.25 8 Vi-TT.sub.ADH Pool 2 58.2 126.23
126.20 13.1 18.50 17.74 9 Vi-CRM.sub.ADH Lot 01/alum 56.4 162.52
98.58 13.4 24.15 22.59 10 Vi-CRM.sub.ADH Lot 03 Pool 2/alum 58.7
98.92 93.22 18.8 24.87 30.55 11 Vi-TT.sub.ADH Pool 1/alum 44.6
191.81 151.56 18.3 36.76 38.83 12 Vi-TT.sub.ADH Pool 2/alum 68.1
202.99 180.09 18.0 24.89 21.73 13 Vi-CRM.sub.ADH Lot 01 65.3 271.48
264.39 16.3 33.38 63.96 14 Vi-CRM.sub.ADH Lot 01 134.5 202.51
234.78 32.2 86.61 64.27 NIH 149.02 24.56
[0202] The results are plotted in FIG. 18.
[0203] Various modifications and variations of the present
disclosure will be apparent to those skilled in the art without
departing from the scope and spirit of the disclosure. Although the
disclosure has been described in connection with specific preferred
embodiments, it should be understood that the claims should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the
disclosure, which are understood by those skilled in the art are
intended to be within the scope of the claims.
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